Hepatocyte Bioreactor System For Long Term Culture of Functional Hepatocyte Spheroids

ABSTRACT

A rotating wall vessel is used as a culture vessel and bioreactor for the cultivation of hepatocytes in the form of spheroids to generate a culture with many properties of the intact liver. These properties include enzyme activity comparable to fresh cells and long-term maintenance of viability and cellular function for periods on the order of months. The cultures may be used to produce hepatocyte products, evaluate metabolism of an agent, propagate Hepatitis C virus and test agents as inhibitors of this virus. Thus, the culture system disclosed herein makes long term functional cultivation of human hepatocytes feasible.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention in the field of biology provides a novel methodand culture system for long term culture of functional hepatocytes byfirst preparing hepatocyte spheroids and inoculating these into aspecialized bioreactor that is a rotating wall vessel wherein the cellsare cultured under conditions that promote three-dimensional aggregationand cellular differentiation.

2. Description of the Background Art

Hepatocyte Spheroids

Landry, J. et al., J. Cell Biol. 101: 914-923 (1985) described thespontaneous formation of spheroidal aggregates when primary rat livercells were incubated on a nonadherent plastic dish. Three distinct cellmorphologies were noted: surface monolayer cells; hepatocytes grouped asislands with deposition of extracellular matrix components; andstructures resembling bile ducts. Tong, J Z, et al., Exp. Cell Res.189:87-92 (1990), studied multicellular spheroids formed from newbornrat liver cells and detected secretion of liver proteins, specificallyalbumin and transferrin, for up to 60 days when the culture medium wassupplemented with dexamethasone, glucagon, insulin, and epidermal growthfactor. These investigators (Tong, J Z, et al., Exp. Cell Res.200:326-332 (1992)) also observed maintenance of liver-specificfunctions in spheroid cultures of adult rat hepatocytes, demonstratingmetabolism of lidocaine by the cytochrome P450 (CYP) enzyme 3A2, for upto 14 days. CYP 1A1 was strongly induced by methylcholanthrene,remaining constant for up to 22 days.

Interestingly, the presence of serum factors inhibited spheroidformation under certain conditions (Koide, N. et al., Exp. Cell Res.186:227-235 (1990)).

One of the present inventors and his colleagues (A. P. Li et al., InVitro Cell. Dev. Biol. 28A:673-677 (1992a)) was the first to reportspheroid formation by human hepatocytes. Their simple, yet proven,method involved seeding 5×10⁶ hepatocytes in culture medium on a 100-mmplastic dish and shaking at 50 rpm overnight. This method caused 90% ofthe cells to aggregate in the form of spheroids, which were shown topossess many of the morphological characteristics of intact liver.

U.S. Pat. No. 5,624,839 disclosed that lipid-bound glycosaminoglycanpromoted spheroid formation.

Cytochrome P450s (CYPs)

CYPs are a family of enzymes, localized to the cytoplasmic side of theendoplasmic reticulum of the liver cell, that catalyze the oxidation oforganic compounds, resulting in increased water solubility whichpromotes excretion from the cell. CYPs are obviously important forprocessing xenobiotics. Table 2 lists a number of CYP enzymes in ratliver that are responsible for metabolism (and detoxification) of anumber of drugs.

Once hepatocytes are isolated from the liver and are grown inconventional primary cultures, the activity of these important enzymesis rapidly lost. This loss is particularly prominent for rat hepatocyteswhich lose 80% of their CYP activity in the first 24 hours of culture(Paine, A J, In: Berry, M N et al. (eds.), The Hepatocyte Review, KluwerAcademic Publishers, Netherlands, pp. 411-420, 2000).

Rotating Wall Culture Vessels

Rotating wall vessels or RWVs are a class of bioreactors developed byand for NASA beginning in about 1990 that were designed to growsuspension cultures of animal cells in a quiescent environment thatsimulates microgravity. RWVs were first described in a number of U.S.patents (U.S. Pat. Nos. 5,026,650; 5,153,131; 5,153,133) assigned toNASA, and thereafter in several additional patents (U.S. Pat. Nos.5,437,998; 5,665,594; 5,702,941) assigned to Synthecon, Inc., who servedas a contractor and licensee of NASA Other patents describe the sameprinciple as the RWV, i.e., horizontal rotation for mixing or suspendingcells in culture medium. With the exception of Ingram et al. (U.S. Pat.No. 5,523,228), however, these patents do not disclose the culture offreely suspended cells. For example, in Rhodes et al. (U.S. Pat. No.5,104,802), cells are confined inside a hollow fiber rotating with theculture vessel. U.S. Pat. No. 6,117,674 described a process forpropagating a pathogen in a three-dimensional tissue mass in RWVculture. The foregoing patents are all incorporated by reference intheir entirety.

During operation, an RWV is completely filled with medium and rotatesabout a horizontal axis. Oxygenation occurs in a bubble-free manner viaa silicone rubber membrane that covers the back wall of the cultivationchamber. Cells are evenly distributed and semi-buoyant duringcultivation, and mixing is accomplished without stirring by end-over-endrotation of the vessel (Schwarz, R P, et al., J. Tiss. Cult. Meth.14:51-58, 1992; Cowger, N L, et al., Biotechnol. Bioeng. 64:14-26,1999).

RWVs have proven beneficial to the cultivation of many cell types fortissue engineering applications. Unlike conventional vessels, a RWVaccommodates three-dimensional (3D) assembly and co-location ofdissimilar cell types in a gently mixed environment. The result is moreextensive 3D growth with increased cell-cell and cell-matrixinteractions and cellular differentiation that more closely resemblesorganized living tissue (Spaulding, G F, et al., J. Cell. Biochem.51:249-251, 1993). These properties of RWVs have been exploited to growand study primary cells from various normal tissues (Goodwin, T J, etal., Proc. Soc. Exp. Biol. Med. 202:181-192, 1993; Freed, L E et al., InVitro Cell. Dev. Biol. 33:381-385, 1997) and cells from tumors. Forexample, aggregates of human prostate tumor cells were moredifferentiated in terms of their growth, morphology, and cytoskeletalprotein expression when cultured in a RWV compared to “control” tumorcells grown in conventional spinner flasks or static cultures (Clejan,S. et al., Biotechnol. Bioeng. 50:587-597, 1996). Khaoustov, V I, etal., In Vitro Cell. Dev. Biol. 35:501-509. 1999) described the cultureof human hepatocytes in an RWV, primarily providing morphologicaldescriptions, though this document disclosed continuous albuminsecretion and urea nitrogen production over a period of 20 days.

Of the existing patent disclosures describing functional hepatocytes invitro, with utility as “artificial livers,” none describe or suggest theuse of freely suspended cells in a rotating bioreactor. For example, Liet al. (U.S. Pat. No. 5,270,192) disclosed a hepatocyte bioreactor inwhich hepatocytes or aggregates are entrapped inside a matrix of glassbeads. In connection with hepatocyte spheroid formation, U.S. Pat. No.5,624,839 (noted above) disclosed a composition that promoted thisprocess.

Citation of the above documents is not intended as an admission that anyof the foregoing is pertinent prior art. All statements as to the dateor representation as to the contents of these documents is based on theinformation available to the applicant and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

SUMMARY OF THE INVENTION

The present invention is directed to the use of a rotating wall vessel(RWV) for the cultivation of hepatocytes in the form of spheroids togenerate cultures that maintain many important properties of intactliver. These properties include long-term viability and maintenance ofcellular function for periods on the order of months, and enzymeactivities comparable to fresh cells. The type of RWV in the presentinvention is termed a High Aspect Ratio Vessel (“HARV”) which isdepicted in the patent cited above and in other related publications.See also, Cowger and O'Connor, 1997, “Application of simulatedmicrogravity to insect-cell culture,” In: Maramorosch, K et al. (eds),Invertebrate Cell Culture, Novel Directions and BiotechnologyApplications, Science Publishers, Enfield, N.H., p. 131-138, herebyincorporated by reference.

The present invention is directed to a method for cultivation ofmammalian hepatocytes in a viable functional state for a prolongedperiod, preferably at least about 7-30 days, which comprises the stepsof:

-   (a) culturing a single cell suspension of mammalian hepatocytes for    a period of between about 12 and about 168 hours, preferably between    about 24 and 72 hours in a flat surface-containing culture vessel    under conditions that permit aggregation of hepatocytes into    spheroids comprising viable cells;-   (b) obtaining the spheroids and introducing them into a rotating    wall vessel culture chamber where the spheroids remain in suspension    during subsequent culture with rotation;-   (c) incubating the vessels under conditions of rotation such that    the spheroids remain in suspension and the hepatocytes remain viable    and capable of metabolizing drugs or toxins via enzymes of the    cytochrome P450 system, which drugs or toxins are ones that are    normally metabolized by hepatocytes in vivo.

Pre-aggregation may be performed in any culture vessel where adherenceto the vessel surface is inhibited or discouraged. Vessels coated withmaterials such as polymethacrylate or poly-L-lysine that inhibitelectrostatic or other interactions that cause cells to adhere may beused. Any form of stirring or mixing, including conventional spinnercultures, may be used at this stage, as long as the conditions promoteaggregation and spheroid formation.

The source of primary hepatocytes may be any of a number of mammalianspecies, preferably human, but also rat, mouse, pig, rabbit, or nonhumanprimate.

The methods disclosed herein for culture and testing of hepatocytes canalso be used with other cell types, particularly cells that do notthrive or function adequately without pre-attachment to other cells orto solid supports. Such cells include primary mammalian cells that donot originate in the liver; and various types of stem or progenitorcells.

The culture medium in the present invention can be any basal medium, orcombination thereof, that supports hepatocyte viability, includingWaymouth MB 752/1; Williams' Medium E; Eagle's MEM; Dulbecco's MEM/Ham'sF12; RPMI1640; or Leibovitz L-15. Preferably, the medium containssupplements that help maintain hepatocyte morphology and differentiatedfunction. These supplements can be conveniently categorized as: (1)growth factors, such as epidermal growth factor (EGF); (2) hormones,preferably peptide hormones such as insulin and glucagon; and (3)glucocorticoids, such as dexamethasone and hydrocortisone.

Any of the known RWVs can be used herein, although a preferred RWV is aHARV.

In another embodiment, this invention provides a method for evaluatingthe metabolism of an agent that is metabolized by mammalian liver cellsin vivo, comprising

-   (a) culturing mammalian hepatocytes as described above;-   (b) adding the agent being evaluated to the hepatocyte culture in    the culture vessels for a period of time sufficient for enzymes of    the hepatocytes to metabolize the agent and converting it to one of    more metabolites thereof;-   (c) identifying the presence of, or measuring the concentration of,    the one or more metabolites in the medium or cells of the culture,    thereby evaluating the metabolism of the agent.

Also included is a method for producing a product, preferably abiomolecule such as a liver protein, e.g., albumin, that is made by,and, optionally, secreted by, normal mammalian hepatocytes in vivo,comprising

-   (a) culturing mammalian hepatocytes as described above, optionally    in the presence of an agent that induces synthesis and/or secretion    of the product, for a period of time sufficient to stimulate the    synthesis and/or secretion of the molecule;-   (b) obtaining the product from the medium or cells of the culture,    thereby producing the product.

Also provided is a method for cultivation of mammalian hepatocytes in aviable functional state, which comprises inoculating a HARV withpre-aggregated hepatocytes or nascent hepatocyte spheroids, in a culturemedium and incubating the hepatocytes and under conditions whereinhepatocytes are viable and metabolically active for a period of at leastabout 4 days, preferably at least about 7 days, more preferably at leastabout 14 days, even more preferably at least about 30 days.

In another embodiment, the present invention is directed to a method forpropagating Hepatitis C virus (HCV) in cultured hepatocytes, comprising:

-   (a) culturing primate hepatocytes as described above;-   (b) inoculating the cultured hepatocytes with an inoculum of HCV,    for example serum from an HCV infected subject, and permitting the    virus to infect, replicate in and be released from the hepatocytes;-   (c) harvesting the medium of the culture which contains the HCV,    thereby growing HCV.

The above method may further comprise the step (d) enriching orisolating the HCV from the medium.

The amount or titer of HCV present in the harvested medium or in theenriched or isolated preparation may be measured, preferably using oneor more of the following criteria: (i) presence of HCV negative-strandRNA; (ii) presence of HCV positive-strand RNA; (iii) transmission ofinfection to cells of a fresh hepatocyte culture by transfer of themedium or enriched/isolated viral preparation; and (iv) infection ofchimpanzees by intravenous inoculation of the medium orisolated/enriched virus, preferably at a dose of at least about 10⁴CID₅₀.

Also provided herein is a method for testing an agent for its activityas an inhibitor of HCV replication or propagation in hepatocytes,comprising:

-   (a) culturing primate, preferably human, hepatocytes in accordance    with claim 1;-   (b) inoculating the cultured hepatocytes with an inoculum of HCV and    permitting the virus to infect, replicate in and be released from    the hepatocytes;-   (c) before, during or after step (b), adding to the virus-infected    cultures the agent being tested for inhibitory activity;-   (d) to a parallel culture or set of cultures of hepatocytes infected    with HCV as in steps (a) and (b), adding negative control agent that    does not inhibit HCV replication or propagation-   (e) harvesting the medium of the cultures of (c) and (d); and-   (f) measuring the amount or titer of HCV present in the harvested    medium,    wherein a lower amount or titer of virus in the cultures of (c)    compared to the cultures of (d) is indicative that the agent is an    inhibitor.

The foregoing method may further include the use of a positive controlgroup in which an agent or combination of agents known to inhibit HCVreplication or propagation is added to a parallel culture or set ofcultures. The amount or titer of resulting virus in the presence of thepositive control inhibitor or inhibitors should be significantly lessthan the amount in the cultures of (d) above, thereby indicating thatthe virus replication or propagation in the cultures is inhibitable bythe positive control agent. The amount of virus in the test group (c)may be higher than, similar to or lower than the positive control,depending on the inhibitory potency of the test agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the novel method for processingprimary hepatocytes and preparing spheroids for inoculation into theHARV bioreactor.

FIGS. 2A and 2B show large viable aggregates composed of multiplehepatocytes in HARV cultures at day 11 (FIG. 2A) and day 16 (FIG. 2B)using the method of the invention. Very few cells in these aggregatesstain with trypan blue (seen as black coloring in these grayscalephotographs), indicating high viability. The overall color of thesespheroid aggregates ranges from tan to dark brown, as would be expectedfrom a large mass of viable liver cells.

FIG. 3 shows a transmission electron micrograph (TEM) of rat hepatocytesfrom HARV culture showing numerous rough endoplasmic reticulum (rer);mitochondria (mi); lysosomes (l); Golgi apparatus (g); membranejunctions (m) with interdigitation of the membranes, and storagevesicles (v) that may be lipid droplets.

FIG. 4 is a graph showing albumin production over time by spheroidscultured in Petri dishes versus spheroids cultured in HARVs.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Following their discovery that single cells suspensions of hepatocytesdo not survive or function adequately following their inoculation intoany of a number of culture vessels or systems, including RWVs, thepresent inventors discovered that pre-aggregation of the hepatocytes fora period of between about 24 hours and 7 days in culture dishes beforetransfer to the RWV permits the hepatocytes to survive and function asnormal liver cells for prolonged periods. This approach and culturesystem permits many practical uses of these long term cultures that haveheretofore not been possible, as described below.

It was initially believed that certain advantages accompanied the use offresh hepatocytes as a single cell suspension to inoculate the RWV.These advantages included saving time and effort compared to“pre-aggregation” (see below) and the potential for a steady maintenanceof enzyme activity. However, experiments conducted with single cellsshowed the rapid loss of viability after several hours in either of twotypes of RWVs, which may be related to inadequate oxygen tension. Inpetri dishes, pO₂ is stable at 130 to 140 mm Hg. In contrast, in theRWV, the pO₂ of oxygen-saturated medium fills rapidly followinginoculation to less than 60 mm Hg.

Hepatocyte spheroids (discussed in more detail below) survive andfunction well in the HARV bioreactor but not in the Slow-Turning LateralVessel (STLV), also known as a Cylindrical Cell Culture Vessel. The HARVis designed with a larger ratio of gas-exchange surface area-to-volumeand thus oxygenates more efficiently than the STLV.

Other factors likely contribute to the fate of single cells inoculatedinto the RWV. The addition of serum to the medium shows potential toimprove the outcome. Higher production of three metabolites ofdextromethorphan were observed when single-cell rat hepatocytes werecultured in medium supplemented with 2% serum. Metabolite production wasenhanced by 40-70% in the presence of serum. After 24 hours, hepatocytescultured as cells in serum-free medium were nonviable whereas, in thesame cultures with serum, approximately 50% of the cells formedaggregates and remained viable.

In view of the foregoing disadvantages of single cell hepatocytesuspensions, the present inventors conceived of, and demonstrated thatpre-aggregation of hepatocytes into spheroids before adding them to theHARV bioreactor resulted in the sought-after characteristics oflong-term hepatocyte cultures, characterized primarily by prolonged cellviability of the culture and normal hepatocyte function. Such longevityof viability and differentiated function may be any period of timeexceeding that which was previously known for hepatocytes, e.g., atleast about 4 days, preferably at least about 7 days, more preferably atleast about 14 days or even more than 30 days.

Thus, the present invention comprises inoculating a HARV withpre-aggregated hepatocytes or nascent spheroids. The procedure forpreparing the spheroids is described in more detailed below and depictedschematically in FIG. 1. Use of pre-aggregated hepatocytes in such abioreactor results not only in successful cultures, but culturespossessing many advantages over other culture types that were known inthe art.

The present bioreactor and culture system has many uses, some of whichare listed and discussed below.

Pathogen Infectivity Studies

Hepatitis C is an important pathogen for which no vaccine is yetavailable. There is no reliable in vitro method for culturing hepatitisviruses and an urgent need in the art for such a capability for thedevelopment of new therapeutics. The present long term hepatocyteculture will permit cultivation of Hepatitis C virus.

Studies of Long-Term Liver Toxicity of Drugs

There is a need for a convenient and reliable laboratory tool to studyliver toxicity of any new drug under development or to evaluate agentswhich are considered to have chronic liver toxicity such as those whoseeffects are manifest only over long periods of exposure in vivo. Thepresent hepatocyte culture system is ideally suited for such studies, asillustrated by the present Examples showing metabolite formation invitro.

Mass Production of Secreted Liver Biomolecules, Particularly Proteinsand Lipids.

Commercial production of biomolecules from hepatocytes is enabled by thebioreactor and culture system of the present invention. Albuminproduction is exemplified below. The present invention is especiallyadvantageous for production of liver-derived compositions that have beendifficult to obtain by other means. The present system is ideal forproduction of bioactive metabolites from prodrugs. Thus, a prodrug typeof compound may be added to the present RWV hepatocyte cultures forlarge scale production of its active metabolite which may be difficultto prepare economically by conventional chemical synthesis. It isbelieved that scale-up to larger volume cultures can be accomplished bythose of ordinary skill in the art without undue experimentation.

Isolation of Hepatocytes

Hepatocytes are isolated from perfused liver using well-establishedmethods, e.g., Li et al., 1992b, J. Tissue Culture Meth. 14:139-146).Likewise, the procedure for preparing hepatocyte aggregates or spheroidshas been published by Li et. al., 1992a, supra and is outlined in FIG.1.

In the present inventors' laboratory, rat hepatocytes cells are commonlyderived from male Sprague-Dawley rats weighing 225-250 g. Porcine,monkey or ape hepatocytes as well as human hepatocytes, which areprepared from donor livers obtained from accident victims or otherrecently deceased donors in whom liver function is believed to benormal, are prepared in a similar manner, though volumes are adjustedaccordingly.

In the “pre-aggregation” or “spheroid-formation” stage of culture, theisolated hepatocytes are cultured in 100-mm diameter sterile petridishes, or an equivalent thereof, with 10 mL of Waymouth's 752/1 medium,pH 7.28, or an equivalent thereof. Medium is preferably supplementedwith the following: 2.24 g/L sodium bicarbonate, 2.38 g/L HEPES buffer,11.2 mg/L alanine, 12.8 mg/L serine, 24 mg/L asparagine, 0.3 mLheptanoic acid, 5 mg/L linoleic acid, 0.175 mg/L aminolevulinic acid, 5mg/L insulin, 5 mg/L transferrin, 5 μg/L selenous acid, 39.2 μg/Ldexamethasone, 0.25 mg/L amphotericin B, 84 mg/L gentamicin sulfate, 84mg/L amikacin sulfate, 100 U/mL penicillin G sodium, and 100 mg/Lstreptomycin sulfate. Those of ordinary skill in the art will know howto select these additives and manipulate their concentrations to achievethe objectives of this invention with ordinary experimentation that isnot undue.

Any conditions that prevent cell adherence to the bottom of the dish (orother surface of alternate vessels) are preferred during this stage.Although cell concentration is typically about 10⁶ cells/mL, and0.5-1×10⁶ is preferred, a broader range of concentrations may be used,for example, between about 10⁴ and 10⁸ cells/mL, as long as the selectedconcentration permits and preferably promotes, spheroid formation.

The preferred bioreactor is a RWV such as the 10-mL HARV produced bySynthecon, Inc. (Houston, Tex.), filled with medium of the abovecomposition (or equivalent) and containing about 5×10⁷ pre-aggregatedrat hepatocytes, though a broader range of cell concentrations may beused. Various drugs or other substrates may be added to the bioreactorand the metabolites collected for analysis of hepatocyte activity as isexemplified below. In a typical HARV, the bottom member of the vessel iscomposed of a paraformaldehyde-based plastic, in a preferred embodiment,Delrin® (or another tough plastic such as that used for molded articles,gears, etc.). A silicone rubber membrane is fixed onto its surface withsilicone sealant. On the vessel's top member, composed of a plasticpolymer such as poly(4,4′-isopropylidine diphenylcarbonate orpoly(4,4′-carbonato-2,2′-diphenylpropane (in one embodiment, Lexan®),are two stainless-steel syringe ports for sampling and feeding. A largerfilling port with a cap is present on preferred larger HARVs, but not onthe 10-mL model. When the vessel's two members are bolted together, achamber is formed—commonly having a 10-mL culture volume that is to becompletely filled with medium during operation. All vessel parts areautoclavable.

As for the rotator base unit, for operation, the vessel is mounted onthe motor shaft. An air pump delivers filtered air to the back of thevessel where gas exchange occurs across the silicone membrane.

Below is an exemplary and preferred procedure for preparing thebioreactor for use, adding substrate, and collecting metabolites. It isunderstood by those of skill in the art the procedures and reagentsdescribed below may be modified by the user in accordance withconventional procedures and knowledge in the art of cell culture anddrug metabolism. For example, if using a larger HARV, volumes, cellnumbers, reagent concentrations, times, etc., may be subject to a rangeof modifications without materially changing the nature of the presentinvention.

Basic Startup Procedure

1. Autoclave a 10-mL HARV according to the manufacturer's instructions,with peripheral screws loosened and foil covering the syringe ports.2. Assemble the vessel inside a sterile hood. Using an Allen wrench,tighten the six peripheral screws. Remove the foil covering the twosyringe ports and install a sterile one-way stopcock valve onto each.Set the vessel aside.3. Obtain the cells to be inoculated in the form of hepatocyte spheroidsprepared in flat surface culture vessels, preferably 100 mm petridishes, from the incubator and transfer to the sterile hood.4. Remove 8 to 9 mL of medium from each dish using a pipet. Cells may beconcentrated from the pre-aggregation cultures by gravity sedimentation(at 1×g) or velocity sedimentation. Simple centrifugation to concentratethe cells is preferably avoided.5. Remove the cells from several (typically 5 to 10) dishes using asingle 10-mL syringe. Attach the syringe onto one of the HARV ports andinoculate the cells into the vessel. Commonly, about 5×10⁷ cells areadded to each 10-mL HARV. During inoculation of the HARV, it isimportant that the aggregates/spheroids be handled gently withminimization of shear forces.6. Discard the inoculation syringe. Fill a new 10-mL syringe withmedium, and attach it to one valve port. Add medium to the vessel untilnearly full while manipulating air bubbles underneath the second openport.7. To remove remaining air bubbles, attach a 5-mL syringe to the otherport and position the vessel so that the bubbles move directly underthat port. Push on the 10-mL inoculating syringe to force the bubbles toescape via the other port and into the 5 mL syringe.8. Once the bubbles have been removed, close the valve, remove anddiscard the 5-mL syringe, but leave the 10-mL syringe, with some mediumremaining, attached.9. Wipe the open port with, e.g., an alcohol or other antiseptic pad,and attach an end cap.10. The vessel can now be placed onto its rotator base inside a 37° C.incubator having a 5% CO₂ atmosphere. The vessel is typically rotated ata speed of approximately 29 rpm. The precise speed is optional and isgoverned by the need to maintain the spheroids in gentle suspension. Asaggregates increase in size over time, the rotational speed may beincreased manually.

Modified Procedure for Adding Substrate

Following Step 6, above:

7A. Remove the one-way stopcock valve from the empty syringe port,keeping it sterile.8A. Add the desired substrate using a micropipettor with sterile tip.Preferably, add no more than 1% (v/v) of any nonaqueous solvent with thesubstrate. Insert the micropipettor tip directly inside the HARV syringeport and add the substrate to the vessel contents. Place the one-waystopcock back onto this port.9A. Attach a 5-mL syringe onto this valve port and remove the 10-mLsyringe from the other side, keeping it sterile. With the 5-mL syringe,gently remove 25 to 50% of the medium, keeping the cells, relativelyundisturbed. Then, gently push these contents back into the vessel. Thisstep insures that the contents are well mixed. Note that a modifiedmixing method may be equally effective.10A. Re-attach the 10-mL syringe. Use the 5-mL syringe to withdraw aninitial sample of about 0.5 mL, replacing that volume with fresh mediumfrom the 10-mL syringe. Store the initial sample frozen until ready toanalyze (e.g., by HPLC).11A. From this point, follow steps 7 through 10 above.Collecting Metabolites from the Bioreactor1. Remove the HARV from its rotating base. Dispose of any extra mediumin the attached 10-mL syringe.2. Using a 10-mL syringe; remove contents (medium plus cells) from thevessel and place into a conical centrifuge tube. At this point, cellviability can be determined using a small sample of the cells.3. Add an equal volume of “stopping” solution to the conical tube andmix. The “stopping” solution may be (i) methanol, (ii) a 1:1methanol/0.1 M Tris acetate mixture, or some other solvent, depending onthe properties of a particular substrate.4. Add 5 mL of “stopping” solution to the HARV and mix it around tocontact all surfaces. Withdraw this solution and save it separately asthe extraction sample. This step is included to increase yield ofsubstrate and/or metabolites, as some compounds have become adsorbed tothe vessel surfaces.5. With the vessel contents mixed with solvent from step 3, centrifugeat 1000 rpm (250×g) or any gravitational force that pellets the cellswithout disrupting them, e.g., from about 150-400×g, for approximately 5minutes.6. Transfer samples of the supernatant to HPLC vials or, alternatively,store frozen until analysis time. The concentrations of substrate andmetabolites derived from HPLC analysis must be corrected for dilution bythe solvent in step 3.7. The cell pellet from step 5 should be processed to (1) recover theintracellular fraction of metabolite and (2) analyze total protein.Resuspend the cell pellet in 0.1 M Tris acetate. Homogenize on ice. Toan aliquot of this homogenate, add an equal volume of “stopping”solution. Place briefly on ice and then centrifuge. Sample thesupernatant and analyze for metabolite by HPLC. The remaining cellhomogenate may be stored frozen until a convenient time to performprotein analysis.Modified Procedure for Collection of Metabolites from ContinuousBioreactor Culture

The above procedure describes harvest of the entire contents of thebioreactor. For continuous production of a secreted metabolite, theprocedure may be modified to harvest only a fraction of the bioreactorcontents. The same cell culture may then be maintained for an extendedperiod and medium sampled repeatedly to test metabolite levels. Theprocedure is essentially as detailed below for changing medium in theRWV. Up to 75% of the conditioned medium may be conveniently removed andreplaced with fresh medium. The conditioned medium may be processed byimmediate freezing, adding solvent, etc., depending on thecharacteristics of the metabolite of interest and the chosen method forpurification and analysis of the metabolite.

Changing Medium in RWV

The following procedure should be performed on the first day followingstartup and every 2 to 6 days afterwards, preferably about every thirdday.

1. Remove the vessel from rotator base and transfer to a sterile hood.2. Discard the medium in the attached 10-mL syringe. Reattach thissyringe to the valve port. Remove the cap from the second valve port,placing it aside on a sterile alcohol pad, and open the valve.3. Tilt the vessel slightly to allow the cells to settle away from bothsyringe ports. Aggregates settle quickly.4. Pull gently on the 10-mL syringe to remove approximately 25 to 75% ofconditioned medium from the vessel (about 2.5 to 7.5 mL). During thisremoval process, the vessel position will need to be adjusted slightlyin order to keep the syringe port in contact with the medium. Discardthe syringe and its contents save supernatant sample for any desiredmeasurements.5. Fill a new 10-mL syringe with medium. Attach this to the syringeport, and gently push fresh medium into the vessel except for the last 2to 3 mL remaining in the syringe.6. Attach a 5-mL syringe to the second valve port. Maneuver the vesselso that air bubbles are positioned directly underneath this port. Pushon the 10-mL syringe to allow bubbles to escape into the 5-mL syringe.7. Close the valve before removing the 5-mL syringe. Swab this port withan alcohol pad and recap it. Wipe any media spills from the surface ofthe vessel with an alcohol pad. The 10-mL syringe stays on the vesselwith the valve open. Place the vessel back on its rotator base insidethe 37° C. incubator.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention, unless specified.

EXAMPLE I Cell Viability and Morphology

As shown in FIG. 2A-2B, HARV cultures of hepatocyte spheroids produceeven larger spheroid aggregates with high viability. The inventorsexamined the relative sizes of spheroids cultured in HARVs versus thosecultured in Petri dishes. From 50 random measurements, the average HARVspheroids were 2.3 times larger than dish spheroids. Only a small numberof peripheral cells are non-viable, as shown in FIG. 2A-2B by theiruptake of trypan blue stain (seen as black spots in these photos). Alsovisible under the microscope is tan to brown pigmentation in the cells(seen here as dark gray patches), presumably reflecting the presence ofthe bile pigment bilirubin. In the present inventors' experience, suchpigmentation is correlated with spheroid viability. Transmissionelectron micrographs of cultures according to this invention showcharacteristics of hepatocytes that are seen in vivo.

In particular, FIG. 3 shows copious rough endoplasmic reticulum,mitochondria, membrane junctions with interdigitations, lysosomes, Golgiapparatus, and storage vesicles (possibly lipid droplets). In othermicrographs (not shown here), structures resembling bile canaliculi withluminal microvilli were observed.

EXAMPLE II Long-Term Maintenance of Hepatocytes

FIG. 2 shows evidence of the capability of the present bioreactor andculture system to maintain hepatocyte viability for several weeks.Hepatocyte spheroids have been viably maintained, with activity of liverenzymes preserved, for over one month. This distinguishes the presentinvention from all other in vitro systems for simulating metabolism of alive mammal with an intact liver.

The first example of preservation of enzyme activity is shown inTable 1. Here, the activity of the cytochrome P450 enzyme 2B1/2 has beenmeasured via the Pentoxyresorufin O-dealkylation (PROD) assay ondisrupted and homogenized rat hepatocytes cultured in a HARV, as dishspheroids (SP), as monolayers (ML), and compared to freshly isolatedcell suspensions (FS).

Freshly isolated rat liver cells exhibited a PROD activity ofapproximately 50 pmol/mg protein. Cells from this same isolation weremaintained as monolayer, dish spheroid, and HARV spheroid cultures formany days and the PROD activity was measured periodically on small cellsamples from these cultures. For monolayers and dish spheroids, thisactivity diminishes after the first day. In HARV culture, by contrast,PROD activity is maintained and even increases over a period of 39 days.Table 1 also shows the maintenance of testosterone 16β-hydroxylation,another CYP 2B1/2 activity, by HARV cultures over a period of 16 days.

In another example of preservation of enzyme activity, human hepatocyteswere maintained in the HARV for 30 days and then “challenged” with adose of 100 μM phenacetin. Within 6 hours of dosing, the metabolitesacetaminophen and acetaminophen sulfate were measured at concentrationsof approximately 250 and 50 nmoles per mg protein, respectively.

TABLE 1 Day FS ML SP HARV PROD activity (pmol/mg protein) 0 48.6 N/A N/AN/A 1 N/A 11.4  25.0  25.0 4 N/A 0.0 1.9 25.3 7 N/A  0.15 0.0 31.6 16N/A nd nd 53.5 39 N/A nd nd 61.1 TST 16β-OH activity (pmol/mg protein) 0590 N/A N/A N/A 1 N/A 300 300 nd 4 N/A  80  0 190 7 N/A 180  0 350 16N/A Nd nd 840 N/A = Not applicable nd = not determined

EXAMPLE III Whole-Cell Drug Metabolism

It is well known in the art that many of the liver-specific enzymefunctions disappear following cultivation of hepatocytes in vitro. Thepresent invention provides an important improvement over the prior art.Table 2 summarizes the metabolism of a variety of drug substrates testedon viable whole liver cells (rat and human hepatocyte cultures fromHARV, spheroids cultured in Petri dishes (SP), and freshly isolated cellsuspensions (FS)) using the present bioreactor system. Activity ofmultiple Cytochrome P450 (CYP) and Phase II conjugation enzymes wasexamined. Substrates were added at a concentration of 100 μM, with theexception of tolbutamide (1 mM), and 7-Hydroxycoumarin (10 M). Activityis measured on whole live cells in culture and analyzed by HPLC of theculture medium.

TABLE 2 nmoles/mg protein/24 hr Metabolites 1-day 7-day 1-day 7-daySubstrate measured Enzyme FS SP SP HARV HARV Tolbutamide 4-OH- 2B; 2C 450 18 14 31 tolbutamide Warfarin 4-OH-warfarin 3A 2.3 43 13 15 2.46-OH-warfarin 1A; 2C 0.44 0.0 2.5 0.05 1.1 7-OH-warfarin 2C 0.05 0.40.77 0.0 0.45 Chlorzoxazone 6-OH- 2E; 1A 4.7 2.7 2.9 1.3 1.1chlorzoxazone Dextromethorphan dextrorphan 2D 5.6 1.5 0.85 0.63 0.333-methoxy- 3A 5.5 7.9 2.7 1.7 9.2 morphinan 3-hydroxy- 2D; 3A 0.99 1.60.3 0.61 1.0 morphinan 7-Hydroxycoumarin 7-HC sulfate GST* 0.41 2.3 1.85.8 5.0 (7-HC) 7-HC UDP-GT* 2.1 2.1 8.3 11 30 glucuronide Midazolam1-OH-midazolam 3A 10 7.3 8.6 1.4 4.5 4-OH-midazolam 3A 33 10 15 2.6 17Acetaminophen (a) a. sulfate GST 7.5 250 130 4.2 30 a. glucuronideUDP-GT 0.87 41 52 11 33 Phenacetin Acetaminophen 1A 0.37 9.2 54 11 50 a.sulfate GST 6.0 34 17 10 6.4 a. glucuronide UDP-GT 0.0 0.0 0.0 0.0 0.0Phenacetin^(†) Acetaminophen 1A nd 200 21 44 70 *Phase II metabolismenzymes: GST = glutathione-S-transferase; UDP-GT = uridine diphosphateglucuronyltransferase ^(†)Second group of phenacetin data applies tometabolism by human hepatocytes. Note that in a separate experiment withhuman hepatocytes in the HARV, a secondary metabolite, acetaminophensulfate was detected.

This drug metabolism is shown in comparison to that measured forhepatocyte spheroids in Petri dishes and freshly isolated hepatocytes insuspension. Table 2 shows the metabolites thus far identified by HPLCassay and the amount produced per day per milligram of cellular protein.These metabolites are the result of action by CYP enzymes, with severaldistinct Phase I oxidation activities measured, and/or conjugation byPhase II enzymes.

With reference to Table 2, each “suspension culture” consisted offreshly isolated liver cells added to “adherent” 24-well plates to formpartially attached cell monolayers over the 24 hour period during whichmetabolism was measured. Spheroid aggregates of fresh cells were formedin mixed Petri dishes. At the end of this formation period, spheroidswere considered “fully developed” and were designated “1-day old.” Atthis time, spheroids were either combined at a higher concentration in afresh Petri dish or were transferred to a HARV for further culture andassay of metabolite production. Separate and parallel cultures ofspheroids in dishes and spheroids in HARVs were maintained and fed for 7days prior to the final assay of drug metabolite production.

In each study represented in Table 2, hepatocytes were “challenged” byadding a drug substrate to the cultures. Metabolites were measured after24 hours incubation. Comparisons were made between (1) freshly isolatedcells; (2) 1-day and 7-day-old spheroids in dishes; and (3) 1-day and7-day-old spheroids in HARVs.

In more than 60% of the cases for HARV spheroids, 7-day-old culturesgenerated more metabolite than did 1-day-old cultures. This was true inonly 40% of cases for spheroids cultured in Petri dishes. This patternis not surprising given the increase over time in PROD and testosterone16-β-hydroxylation activities documented in Example II for thehepatocyte bioreactor cultures. Like the data in Example II, CYP 2Bactivity is seen again here as well preserved in HARV cultures.

There was an indication of reduced recovery of some substrate andmetabolite compounds from the HARVs, compared with the other culturemodes. This could potentially be due to the following: (1) reducedavailability through adsorption to the bioreactor surfaces or diffusionlimitations due to the larger size of spheroids in the HARV; (2)inadequate recovery of the intracellular fraction of the drug; (3) lossof cellular protein on harvest or feeding. Regardless, the presentsystem exhibits consistent and continuous production of metabolites frommany important drug substrates, as shown. Coupled with the greatlyenhanced longevity of these HARV cultures, the present hepatocytebioreactor system promises to be an excellent tool for drug metabolismstudies.

EXAMPLE IV Protein and Urea Production

The production of albumin, an important plasma protein synthesized inthe liver, was measured in HARV cultures compared to spheroids in Petridishes (FIG. 4) over the course of several days. Albumin production was,on average, 30% higher in the HARV cultures and was maintained for atleast 11 days.

The inventors measured endogenous urea production by rat hepatocytes asspheroid cultures in HARVs, spheroids in dishes, and monolayers. Theaverage production rates over 11 days in culture were:

Monolayer 35 ± 11 μg urea/mg protein/day Dish spheroids 41 ± 13 μgurea/mg protein/day HARV spheroids 59 ± 6  μg urea/mg protein/dayThe formation of urea from an exogenous source of ammonia was measuredusing porcine hepatocytes in a perfused RWV, a vessel related to,although different from, the HARV. Approximately 80 μmoles of urea wasformed overnight in culture from 170 μmoles of NH₄Cl added.

EXAMPLE V Propagation of Hepatitis C Virus and Drug Testing

To test the ability of the present hepatocyte bioreactor system tosupport infection and propagation of Hepatitis C virus, a culture ofprimary hepatocytes is first established in HARV bioreactors. HepatitisC virus is introduced in the form of infectious serum (from anHCV-infected subject).

The establishment and progress of HCV infection is monitored withregular sampling of cells and/or medium from the bioreactor. Asuccessful outcome is demonstrated by (i) presence of HCVnegative-strand RNA; (ii) long-term production of HCV positive-strandRNA; and (iii) transmission of infection to cells of a fresh hepatocyteculture.

Quantification of viremia is accomplished by detecting viral coreantigen by fluorescent enzyme immunoassay (Tanaka, T et al., 1995, J.Hepatology 23:742-745) by measuring HCV RNA using the Amplicor HCVMonitor test (Roche Molecular Systems, Pleasanton, Calif.) or theQuantiplex HCV RNA 2.0 assay (bDNA; Chiron, Emeryville, Calif.).

Total RNA is purified using the RNAqueous® kit (Ambion, Austin, Tex.) orHigh Pure RNA Isolation kit (Boehringer Mannheim, Germany).

Production of the negative RNA strand is measured by the method oftagged RT-PCR amplification (Lanford, R E et al., 1994, Virology 202:606-614), followed by Southern blot hybridization.

Human infectious serum and human hepatocytes are obtained from reliablesources, with donor consent.

A form of positive control is the passage of viral infection from thesupernatant of one bioreactor to a naïve culture. As a negative control,normal non-infectious human serum is added to identical bioreactorcultures.

The following parameters are varied in these studies: (1) initial numberof hepatocytes in the bioreactor: between 5×10⁶ and 5×10⁸ cells; (2)hepatocyte source: primary or cryopreserved hepatocytes from humanadults or fetuses or from chimpanzees; (3) age of hepatocyte culture(e.g. 1-10 days in bioreactor prior to infection); (4) HCV titer ofinfectious serum, between about 5×10⁶ and about 10⁹ equivalents/mL; and(5) other properties of the serum including titers of anti-HCVantibodies that are specific for HCV proteins.

Bioreactor cultures will be started, initially using the preferred cellconcentration of 5×10⁶ cells/mL. Hepatitis C virus will be seeded intoHARV cultures between days 1 to 10, at titers of 0.25 to 2.5×10⁵copies/mL (Rumin, S. et al., 1999, J. Gen. Virol. 80:3007-3018;Fournier, C. et al., 1998, J. Gen. Virol. 79:2367-2374). Infection issustained, along with the hepatocyte culture, for an extended period,ranging between about 7 and about 30 days, to reach a titer of 0.5 to5×10⁶ copies/mL.

Proof that HCV was successfully propagated is obtained by measuringincreased viral RNA content and by transmission of virus from aninfected culture to a naïve HARV culture where it again propagates.

The ultimate test of successful production of competent infectiousparticles is by infection and Hepatitis C disease induction in achimpanzee model using virus generated in the present bioreactorculture. Using culture supernatants that are positive by in vitroinfectivity, quantities equivalent to 10⁴ to 10^(6.2) 50% chimpanzeeinfective doses (CID₅₀) will be administered i.v. to chimpanzees(Feinstone, S M et al., 1983, Infect. Immun. 41:816-821; Shimizu, Y K etal., 1990, Proc. Nat. Acad. Sci. 87:6441-6444). Clinical signs of acutehepatitis C will appear in 80% of the recipients within 6 to 15 weeks.These signs include elevated serum alanine aminotransferase levels,anti-HCV antibodies, serum viral RNA measurable by qualitative PCR,and/or ultrastructural changes seen on liver biopsy.

The foregoing Hepatitis C model is used to test the effect of atherapeutic candidate, whether small molecule, peptide or biologicalmacromolecule. Different concentrations of the candidate in parallelwith a negative control, are added to cultures established underselected conditions determined as above. The cultures are monitored fora decrease in viral load or a static effect on viral growth incomparison with the negative control. As a positive control, an agent orregimen with known anti-HCV activity can be run concurrently, forexample, the nucleoside analogue Ribavirin (e.g., Virazole®, Rebetol®),interferon-α, preferably interferon α-2b (e.g., Roferon-A®, Intron-A®)(which is the FDA-approved standard treatment of HCV infection), or thecombination of Ribavirin and interferon-α.

The present bioreactor system may be used as a model for infectiousdiseases other than Hepatitis C, wherein the pathogenic organismreplicates in hepatocytes, with minor modifications to the protocol thatare tailored to the pathogenic organism of interest.

The references cited above are all incorporated by reference herein,whether specifically incorporated or not.

Having now fully described this invention, it will be appreciated bythose skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations, and conditions withoutdeparting, from the spirit and scope of the invention and without undueexperimentation.

1-29. (canceled)
 30. A method for testing an agent for its activity asan inhibitor of HCV replication or propagation in hepatocytes,comprising: (a) preparing cultured hepatocytes by (i) culturing a singlecell suspension of mammalian hepatocytes in a culture medium for aperiod of between about 12 and about 168 hours in a culture vesselhaving a flat surface under conditions that permit aggregation ofhepatocytes into spheroids which comprise viable hepatocytes; (ii)transferring said spheroids into a rotating wall vessel (RVW) culturechamber where said spheroids remain substantially in suspension duringsubsequent culture with rotation; (iii) incubating said suspendedspheroids under conditions of vessel rotation such that said spheroidsremain substantially in suspension; wherein suspended spheroids remainviable and capable of: (1) producing and secreting normal hepatocyteproducts; and (2) metabolizing drugs or toxins via enzymes of thecytochrome P450 system, which drugs or toxins are ones that are normallymetabolized by hepatocytes in vivo for more than 30 days; (b)inoculating the cultured hepatocytes with an inoculum of Hepatitis CVirus (HCV) to prepare virus-infected cultures and permitting the virusto infect, replicate in and be released from the hepatocytes; (c)before, during or after step (b), adding to the virus-infected culturesthe agent being tested for inhibitory activity; (d) to a parallelhepatocyte culture or set of cultures of hepatocytes infected with HCVas in steps (a) and (b), adding negative control agent that does notinhibit HCV replication or propagation (e) harvesting the medium of thecultures of (c) and (d); and (f) measuring the amount or titer of HCVpresent in the harvested medium, wherein a lower amount or titer ofvirus in the cultures of (c) compared to the cultures of (d) isindicative that the agent is an inhibitor.